Molecular searches use one of several forms of complementarity to identify the macromolecules of interest among a large number of other molecules. Complementarity is the sequence-specific or shaped-specific molecular recognition that occurs when two molecules bind together. Complementary between a probe molecule and a target molecule can result in the formation of a probe-target complex. This complex can then be located if the probe molecules are tagged with a detectible entity such as a chromophore, fluorophore, radioactivity, or an enzyme. There are several types of hybrid molecular complexes that can exist. A single-stranded DNA (ssDNA) probe molecule can form a double-stranded, base pair hybrid with an ssDNA target if the probe sequence is the reverse complement of the target sequence. An ssDNA probe molecule can form a double-stranded, base-paired hybrid with an RNA target if the probe sequence is the reverse complement of the target sequence. An antibody probe molecule can form a complex with a target protein molecule if the antibody's antigen-binding site can bind to an epitope on the target protein. There are two important features of hybridization reactions. First, the hybridization reactions are specific in that the probes will only bind to targets with a complementary sequence, or in the case of proteins, sites with the correct three-dimensional shape. Second, hybridization reactions will occur in the presence of large quantities of molecules similar but not identical to the target. A probe can find one molecule of a target in a mixture of a zillion of related but non-complementary molecules.
There are many research and commercially available protocols and devices that use hybridization reactions and employ some similar experimental steps. For example, microarray (or DNA chip) based hybridization uses various probes which enable the simultaneous analysis of thousands of sequences of DNA for genetic and genomic research and for diagnosis. Most DNA microarray fabrications employ a similar experimental approach. The probe DNA with a defined identity is immobilized onto a solid medium. The probe is then allowed to hybridize with solutions of nucleic acid sequences, or conjugates, that contain a detectable label. The signal is then detected and analyzed. Variations of this approach are available for RNA-DNA and protein-protein hybridizations and those hybridization techniques involving tissue sections that are immobilized on a substrate. In all of these protocols, the hybridization solution is placed directly on the substrate that contains the immobilized DNA or tissue section.
Usually the hybridization reaction is performed in a warm environment, and one problem is that the hybridization solution may evaporate resulting in inadvertent contamination of the solution that is on the substrate. To prevent evaporation, a cover slip is placed directly on the solution. The weight of the cover slip, however, can displace the solution and minimize the volume of the solution that is in contact with the immobilized component. Therefore, a portion of the immobilized component may have an insufficient amount or no hybridization solution covering it.
In an attempt to reduce the displacement of the cover slip, it is known to print ink as bars on two opposed edges of the cover slip. The ink structures raise the cover slip above the substrate thus providing a chamber and a larger volume or space for the hybridization solutions. However, the cover slip is often made from a #2 cover glass that has a nominal thickness of about 0.2 millimeters (“mm”). As will be appreciated, a nominal thickness of 0.2 mm means that, if measured, the cover glass thickness may vary in a range of about 0.17-0.25 mm. Further, the dimensional range of cover glass also varies with the supplier; and thus, a variation in the measured dimension from the nominal dimension is to be expected. A cover glass that is 0.2 mm thick is thin and flexible. Further, the cover slip is readily flexed or bent by the application of a very small force. For example, in reacting adhesion forces resulting from the hybridization solution being introduced between the coverslip and the substrate, a center portion of the cover slip between the printed ink bars can be warped or pulled toward the substrate. Once again, the hybridization solution is displaced resulting in an unequal dispersion of the components in the solution over the immobilized component. Alternatively, in filling the space between the cover slip and the substrate with hybridization solution, the cover slip can be warped or pushed outward. Pushing the cover slip outward increases the volume of the chamber. Under other circumstances, the very thin cover slip can unpredictably warp or bend due to forces inherent within the structure of the glass or from forces generated by the printing and curing of the ink bars on the cover slip.
Regardless of its cause, any bending, deflecting or warping of the cover slip results in different distances between the cover slip and the substrate; and that warping and bending of the cover slip can cause substantial variations in the size of the chamber between the cover slip and the substrate. One disadvantage of varying spacing between the cover slip and the substrate is that different amounts of hybridization solution are dispersed to different areas over the immobilized component, thereby producing a less consistent hybridization reaction and less consistent hybridization results. Another disadvantage of varying spacing between the cover slip and the substrate is that greater volumes of hybridization solutions will be used. Using more than the minimum necessary adds unnecessary cost to the hybridization reaction. It has been found that with the opposed printed bars on the cover slip, evaporation of the hybridization solution continues to be a problem. In addition, the bending and/or warping of a cover slip results in a varying distance between the cover slip and substrate from one cover slip/substrate combination to another. Again, that nonuniformity between cover slip/substrate combinations often leads to inconsistent results as well as an increased process cost due to the use of more hybridization solution.
Thus, there is a need for a cover slip for use with a substrate that provides more consistent results from a hybridization reaction or similar molecular search, uses a consistent and minimum amount of hybridization solution or other liquid, and further reduces evaporation.